Composition of matter for inhibiting leukemias and sarcomas

ABSTRACT

A new antineoplastic substance denominated &#34;pancratistatin&#34;. Methods of isolating natural and synthetic pancratistatin and 7-deoxynarciclasine and the pharmaceutically acceptable derivatives thereof to make a pharmaceutical preparation therefrom and methods for treating a host afflicted with a neoplasm with said preparations.

INTRODUCTION

The present invention relates generally to a new antineoplasticsubstance denominated "pancratistatin", its synthetic counterpart andpharmaceutically acceptable derivatives thereof. The invention furthercomprises methods for obtaining pancratistatin and 7-deoxynarciclasine,proceedures for creating a pharmacutical preparation therefrom, andmethods for treating a host, afflicted with a neoplastic growth,therewith.

BACKGROUND OF INVENTION

As early as the fourth century BC, attention was directed to certainmedical and/or poisonous plant species. To determine the presence ofalkaloid constituents in such species, see: Gibbs, R.D. "Chemotaxonomyof Flowering Plants" Vol III, McGills-Queens University Press. Montreal,1974, p 1924. Over time, more than thirty species of the relativelylarge Amaryllidaceae family found use in the primitive treatment ofcancer (See: Hartwell, J. L. Loydia, 1967, 30, 391).

In the continuing effort to locate and define various natural andsynthesizable substances for treatment of one or more varieties ofcancer, research chemists continue to look at natural flora and fauna inan attempt to isolate and identify substances which exhibitantineoplastic activity while substantially minimizing, if not totallyeliminating, some of the severe side effects accompanying knownchemotherapeutic agents.

It is in the further pursuit of these goals that plant speciesheretofore ignored are now being examined to determine whether theycontain constituents which when isolated will exhibit antineoplasticactivity.

Accordingly, a principal object of the present invention is to providenew agents useful in the retardation or remission of one or more typesof cancer.

Another object of the present invention is to provide methods andprocedures for isolating antineoplastic substances from plant life in aform whereby they may be readily and usefully employed in thetherapeutic treatment and management of one or more types of canceroccuring in human hosts.

A further object of the present invention is to provide means andmethods of creating useful pharmaceutical preparations for the treatmentand management of cancer which preparations contain as their essentialactive ingredient a factor extracted from Pancratium littorale Jacq. andZephranthes grandiflora, its synthetic replication, or a non-toxicpharmacologically active derivatives thereof.

These and still further objects, as shall hereinafter appear, arefulfilled by the present invention in a remarkably unexpected manner aswill be readily discerned from the following detailed description ofexemplary embodiments thereof.

BRIEF SUMMARY OF THE INVENTION

The present invention comprises the provision of new and usefulchemotherapeutic agents which are extracted from the root of theHawaiian (or African) Pancratium littorale Jacq. and Zephranthesgrandiflora in the manner hereinafter described in detail, and which arethereafter formulated into useful pharmaceutical preparations havingdemonstratable and confirmed levels of anticancer activity when measuredby the generally accepted protocols in use at the United States NationalCancer Institute. The principal substance above referred to is hereindenominated "Pancratistatin". The invention further contemplates thepreparation of a synthetic counterpart and the non-toxicpharmacologically active derivative of pancratistatin and itspharmcologically active companion, 7-deoxynarciclasine.

One principal active ingredient of the present invention is a substancedenominated pancratistatin having the structure: ##STR1## wherein R═R₁═R₂ ═R₃ ═R₄ ═H, the derivatives thereof wherein R═R₁ ═R₂ ═R₃ ═R₄ ═COCH₃and wherein R═CH₃ and R₁ ═R₂ ═R₃ ═R₄ ═H.

A second active ingredient of the present invention is a substancedenominated 7-deoxynarciclasine having the structure: ##STR2## R₁ ═R₂═R₃ ═H,

DETAILED DESCRIPTION OF EMBODIMENTS

The bulb section (45 kg) of P. littorale was extracted with methylenechloride-methanol-water and pancratistatin was concentrated (separationwas guided primarily by bioassay using the PS in vivo system) in an-butanol extract of the aqueous phase. Purification of half of thecrude product employing selective solubility properties and gelpermeation chromatography (SEPHADEX ® LH-20) afforded 6.5 g (0.028%yield) of pancratistatin that separated fromdimethylformanidemethanol-ether as a colorless solid: dp 320°-321°; EImass spectrum m/e 325 (M⁺, C₁₄ H₁₅ NO₈) [α]³² +44° (c, 1.0, DMSO);CH₃.sbsp.max OH (log Σ) 229 (4.29), 239 (4.26) and 281 (4.0) nm; IR(KBr) ν_(max) 3500-3200, 1675, 1615, 1600, 1500, 1465, 1445, 1420, 1375,1350, 1300, 1230, 1200, 1160, 1118, 1085, 1070, 1040, 1030, 930, 912,880, 840, 720, 655, 640 and 610 cm⁻¹ ; and ¹ H NMR (100 MHz, DMSO-d₆) δ3.6-4.4 (6H), 4.72-5.70 (5H, removed by D₂ O, 6.11 (2H, br. s), 6.56(1H, s) and 13.15 (1H, removed by D₂ O). Reaction of pancratistatin withacetic anhydridepyridine provided its pentaacetate (mp 162°-166°).Reaction of pancratistatin with diazomethane in methanol yielded the7-monomethyl ether (mp 294°-298°). The same procedure providespancratistatin from Zephranthes grandiflora although P. littorale J. isused herein as exemplarly.

The remarkable insolubility of pancratistatin in a variety of organicsolvents, its high decomposition point, non-basic character, andinfrared spectrum suggests a carbostyril or isocarbostyril system. Themost plausible interpretation of the elemental analyses and spectraldata for pancratistatin, the pentaacetate and the methyl ether point toa new phenanthridone. An x-ray crystal structure determination wasutilized to make the stereochemical assignments and confirm the overallstructure of pancratistatin as shown below. ##STR3## wherein: R═R₁ ═R₂═R₃ ═R₄ ═H.

A single crystal (0.125×0.25×0.37 mm) of pancratistatin monomethylether, recrystallized from 95% ethyl alcohol, was found to correspondwith C₁₅ H₁₇ NO₈.H₂ O, fω 357.32; monoclinic B=99.78 (2)₃ °, a=9.040(1),b=8.317(1) and c=10.187(2) Å; V=754.8 Å; F (000) =376; Po=1.565 g/cc,Z=2, and Pc=1.572 g/cc (25° C.; CuKα, λ=1.54184 Å).

Systematic extinctions (OkO absent if k=2n+1) and chirality wereconsistent with space group P2₁. The observed density of 1.56 g/ccindicated one molecular each of C₁₅ H₁₇ NO₈ and water per equivalentposition (2 molecules of pancratistatin /cell). The cell parameters wereobtained by a least-squares fit of the reciprocal lattice positions of25 diffractometer measured reflections with 2-theta values in the rangeof 5°-35°. Intensities of all unique reflections with 2θ<75° weremeasured at 25° C. using a variable speed omega/2-theta scan techniqueon an Enraf-Nonius (Delft) CAD4 diffractometer employinggraphite-monochromated CuKα radiation. The scan angle was calculated foreach reflection as (0.90+0.15 tan θ)°. The detector aperature with avariable width of (4.0+0.5 tan 2) mm and a constant vertical height of 4mm was positioned at a distance of 173 mm from the crystal. Maximum scantime for each reflection was 1 min. Two-thirds of this time was used toscan the peak and one-sixth in measuring ach of the two backgrounds. Theintensities of three monitor reflections were also recorded ever 250minutes and found to have varied by less than 0.5% during the entiredata collection process. A total of 1647 unique reflections wereretained for subsequent processing [I<1.0σ (1)]. All intensity data werecorrected for anisotropic decay using the monitor intensities of thestandards. The data were corrected for Lorentz-polarization andanomalous dispersion effects. Atomic scattering factor coefficients weretaken from appropriate tables: D. T. Cromer and J. T. Waber,"International Tables for X-ray Crystallography," Vol. IV, The KynochPress, Birmingham, England, 1974, Table 2.2B; tables for anomalousdispersion coefficients, D. T. Cramer, ibid., Table 2.3.1. No extinctionor absorption adjustments were made or deemed necessary (μ=10.038 cm⁻¹).

Structural determination of pancratistatin monomethyl ether was achievedby direct methods using a multisolution weighted tangent formulaapproach, i.e., program MULTAN. All crystallographic calculations wereaccomplished with a PDP 11/23 computer using the Enraf-Nonius (Delft)Structure Determination Package (SDP-PLUS) software system developed byB. A. Frenz and Associates, Inc., College Station, Texas, 1982.Principal programs used were; START, PAINTER, REJECT, CHORT, datareduction programs; LSB, a full matrix last-squares refinement program;SEARCH, a connectivity peak search peak program; ORTEP-II thecrystallographic illustration programs; MULTAN 11/82 "A System ofComputer Programs for the Automatic Solution of Crystal Structures fromX-ray Diffraction Data," a direct methods program by P. Main andcolleagues, University of York, York, England. For a description ofMULTAN see: Germain, G; Main, P.; Woolfson, M. M.; Acta Crystallogr.Sect. B., 1970, 26, 274-285 and Woolfson, M. M. Acta Crystallogr. Sect.B., 1977, 33, 219-225. After the correct assignment of co-ordinates forall non-hydrogen atoms, full matrix least-squares refinement thenon-Poisson contribution weighting scheme was used in least-squaresrefinement, where W=1/σ.sub.(F 2.sub.) =4×F.sup. 2 /(∝_(F) 2)² and∝.sub.(F².sub.) =√∝I² +(P×F²)², where P is an adjustment factor todowngrade intense reflections and W is the weight for the reflection. Avalue of 0.05 was used for P on all co-ordinates, scale factor andisotropic temperature factors resulted in a rapid reduction in theconventional unweighted crystallographic discrepancy indexR(Σ|Fo-Fc|/Σ|Fo|) to 0.1795 [for observed reflections, I>3σ (I)].Subsequent difference electron density syntheses revealed all hydrogenatoms as well as the presence of one molecule of water, which appear tobe hydrogen bonded (1.83 Å) to one of the hydroxy hydrogens at C-4.Finally, several additional cycles of full-matrix least-squaresrefinement of all variables (anisotropic nonhydrogen atoms and isotropicnonhydrogen atoms and isotropic hydrogens, with the thermal B values ofthe latter fixed at a nominal value of 5.0) caused convergence toR=0.405 (Rω=0.0438. Refinement was discontinued when parameter shiftsbecame insignificant (maximum shift to error ratio was 0.84 with themajority in 0.1-0.2 range) and a final difference map revealednegligible electron densities (>0.22 e Å⁻³). All bond lengths and anglesagreed with expected values.

A calculation of the discrepancy indexes for the opposite enantiomorphof that depicted for the pancratistatin derivative (see Table I) whenR═CH₃ and R₁ ═R₂ ═R₃ ═R₄ ═H, from the data (1632 observations and 301variables) yielded values of R' =0.0425 and R'ω represents the absolutesterochemical configuration with >99.9% probability. Additional supportfor the absolute configurational assignment arises from a combination ofbiosynthetic and x-ray crystal structure studies of narciclasine, a1-dehydrophenanthridone derivative of pancrastistatin.

In one practice of the present invention, the bulbs of P. littorale wereextracted employing a methylene chloride-methanol procedure followed bythe addition of water. The methylene chloride phase was partitionedusing the solvent sequence 9:1→4:1→3:2 methanol-water with hexane→carbontetrachloride→methylene chloride. The aqueous phase was extracted withn-butanol and pancratistatin was concentrated there rather than in themethylene chloride residue where lycorine would be expected.

The n-butanol fraction was further separated by gel permeationchromatography on SEPHADEX® LH-20 using methanol as eluent. Fractioncollection was guided by thin layer chromatography using 3:1chloroform-methanol. The two principal antineoplastic componentscorresponded to R_(f) 0.37 and 0.46. Both proved to be very highmelting, relatively insoluble, and nitrogen-containing non-basic solidsreminiscent of carbostyrils or isocarbostyrils.

Purification of the R_(f) 0.37 product by recrystallization from aceticacid-methanol afforded a lactam that readily gave a triacetatederivative. Spectral data indicated that this anticancer (PS, T/C 161 at12.5 mg/kg, ED₅₀, 0.02 μg/ml) constituent was a 7-deoxy derivative ofnarciclasine. But physical constants reported for margetine, laterrenamed lycoricidine, indicated otherwise. For these reasons and becauseinitial attempts to obtain an authentic sample of 7-deoxynarciclasinewere unsuccessful, a complete structural determination was undertaken byx-ray crystallographic methods. The result was definite assignment ofstructure ##STR4## to the substance with R_(f) 0.37. The absoluteconfiguration of the triol was deduced from its presumed biosynthesisfrom vittatine. Later authentic samples of the triacetate ##STR5##prepared from the natural and (±) synthetic products were obtained andinfrared spectral comparison in potassium bromide with the substancewith R_(f) 0.37 P. littorale showed substantial differences, but whencompared in a chloroform solution, the spectra were identical. On thisbasis the assignment of the above shown structure to 7-deoxynarciclasineis believed correct.

Recrystallization of the R_(f) 0.46 anticancer (T/C 138→165 at 0.75→6.0mg/kg dose levels and 206 at 12.5 mg/kg in the PS system with ED₅₀, 0.01μg/ml) component from dimethylformamide-methanol-ether gave a purespecimen that displayed elemental analytical and spectral data in accordwith the structure: ##STR6## This new phenanathridone was denominated"pancratistatin" and its structural elucidation was accomplised by anx-ray crystal structure determination. The structure determined forpancratistatin was completely consistent with the corresponding spectralresults.

To further aid in the understanding of the present invention, attentionwill now be directed to the procedures practiced in isolatingpancratistatin and 7-deoxynnarciclasine from Hawaiian (African)Pancraticm littorale Jacq.

General Method. All solvents employed for chromatography wereredistilled. Thin layer chromatography was performed on silica gel GHLFUniplates (0.25 mm layer thickness) supplied by Analtech Inc.,developing solvent chloroform:methanol (3:1) and visualized with cericsulfate spray reagent. SEPHADEX® LH-20 (particle size 25-100 μ) wassupplied by Sigma Chemical Co. A Gilson Model FC-200K fraction collectorwas used to collect fractions.

Melting points were determined on a Kofler-type hotstage apparatus andare uncorrected. Optical rotations were measured with a Perkin-ElmerModel 241 Automatic Polarimeter. Ultraviolet spectra were recorded on aHewlett-Packard Model 8450A UV/VIS spectrophotometer and infraredspectra on Perkin Elmer Model 200 and Nicolet MX-1 FTIRspectrophotometers. ¹ H-and ¹³ C-NMR spectra were recorded on VarianXL-100 and Bruker HXE-90 (22.63 MHz) spectrometers respectively, usingtetramethylsilane as internal standard. Mass spectra were obtained on avarian MAT 312 spectrometer.

Extraction. The bulbs of the Hawaiian P. littorale Jacq were chopped andthe chopped bulbs (45 kg) were extracted with methanol:methylenecholoride (1:1, 320 liters) at ambient temperature for 20 days. Theextract was then decanted and the methylene chloride phase separated bythe addition of 20% by volume of water. The aqueous phase was adjustedby addition of further methanol and methylene chloride in the ratio,aqueous phase:methanol:methylene chloride (2:1:1) and the bulbsre-extracted for a further 20 days. Subsequent decantation and additionof 20% by volume of water separated the methylene chloride phase whichwas then combined with the first methylene chloride fraction andevaporated to give an inactive extract (812 g).

Solvent Partition. The aqueous phase from the above extraction wasconcentrated to approximately 16 liters and thereafter centrifuged toremove the insoluble material therefrom. The clear solution was thenextracted with n-butanol (3×10 liters) and the butanol extractconcentrated to give the butanol soluble fraction (705 g). An aliquot(355 g) was dissolved in methanol (1.5 liter) and acetone (3.5 liters)added. The insoluble material (105 g) was filtered and the filtrateevaporated to provide a residue (250 g).

Isolation of 7-Deoxynarciclasine and Pancratistatin. The residueobtained by the foregoing solvent partition was treated with methanol(1.5 liters) and thereafter filtered to produce a solid (2 g) shown tobe pancratistatin. The filtrate was chromatographed on Sephadex LH-20 (2kg; 105×10 cm) using methanol as eluent and monitoring the fractions bythin layer chromatography. Fractions containing the component r_(f) 0.37were combined, concentrated and filtered to provide 7-deoxynarciclasine(10 g) which crystallized from acetic acid-methanol as fine needles, mp251°-252° [lit. 214.5°-2.15.5° (9); 230° (dec) (12)]; [α]³³ +157.3° (c0.96, DMSO); ei ms: m/e 291 (M⁺, C₁₄ H₁₃ NO₆); λ_(max) ^(CH).sbsp.3^(OH)(log Σ) 233(4.14), 248 (4.15) and 302 (3.75)nm; ir (KBr) ν_(max) 3450,3250, 1672, 1632, 1620, 1602, 1505, 1473, 1415, 1400, 1340, 1320, 1270,1250, 1080, 1045, 1015, 976, 940, 890, 860, 785, 700, 670 and 623 cm⁻¹ ;¹ H nmr (pyridine-d₅) δ, 4.81-4.92 (2H), 5.0-5.35 (2H), 6.05 (2H, d,J=a3 Hz), 6.72 (1H, br s), 7.33 (1H, s), 8.06 (1H, s), 8.57 (1H, br s,removed by D exchange) and 7.0-8.6 (br hump, removed by D₂ O) ppm; ¹³² Cnmr (DMSO-d₆) δ163.12, 150,58, 147.72, 131.70, 130.01, 123.61, 121.95,106.19, 103.24, 101.81, 72.56, 69.21 and 52.74 ppm (one carbon masked byDMSO: 42.77-36.72).

Anal calcd for C₁₄ H₁₃ NO₆ : C, 57.73; H, 4.47; N, 4.81. Found: C,57.79; H, 4.49; N, 4.79.

Further elution gave fractions containing mainly the component R_(f)0.46 which, on concentration and filtration, gave pancratistatin (4.5g). Crystallization from dimethylformamide-methanol-ether gave acolorless solid, mp 320°-321° (dec); [α]_(D) ³² +44° (c 1.0, DMSO) eimsm/e 325 (M⁺,C₁₄ H₁₅ NO₈); λ_(max) ^(CH).sbsp.3^(OH) (log Σ) 209(sh),219(sh), 233(4.32), 278(3.91) and 308 (br. sh)nm; ir (KBr) ν_(max)3500-3200, 1675, 1615, 1600, 1500, 1465, 1445, 1420, 1375, 1350, 1300,1230, 1200, 1160, 1118, 1085, 1070, 1040, 1030, 930, 912, 880, 840, 720,655, 640 and 610 cm⁻¹ ; ¹ H nmr (MDSO-d₆) δ 3.6-4.4 (6H), 4.72-5.70 (5H,removed by D₂ O), 6.11 (2H, br. s), 6.56 (1H, s) and 13.15 (1H, removedby D₂ O) ppm; ¹³ C nmr (DMSO-d₆) δ 169.49, 152.04, 145.38, 135.63,131.70, 107.49, 101.70, 97.62, 73.28, 70.19, 69.99, 68.50 and 50.46 ppm(one carbon masked by DMSO:42.27-36.72).

Anal calcd for C₁₄ H₁₅ NO₈ : C, 51.69; H, 4.61; N, 4.31. Found: C,51.65; H, 4.55; N, 4.24.

Biologically active glycoside derivatives of pancratistatin and7-deoxynarciclasine are made by coupling the principal with suitablyprotected sugars or other hydroxylated compounds using methods wellknown in the art [see Methods in Chemistry by R. L. Whistler and J. N.Bemiller (eds.), Academic Press, N.Y., 1972, Vol. 6, or TheCarbohydrates: Chemistry and Biochemistry by W. Pigman, Academic Press,N.Y., 1981].

Derivatives of pancratistatin are used for the same purposes aspancratistatin.

Pancratistin and 7-deoxynarciclasine, as demonstrated, both have freehydroxyl groups available for derivatization. Thus, acyl esters of thesecompounds can also be prepared by methods well known to those skilled inthe art. Acyl derivatives of the pancratistatins and7-deoxynarciclasines can be used for the same biological purposes as theparent compounds.

Acids which can be used in the acylation of a pancratistatin include:

(a) saturated or unsaturated, straight or branched chain aliphaticcarboxylic acids, for example, acetic, propionic, butyric, isobutyric,tert-butylacetic, valeric, isovaleric, caproic, caprylic, decanoic,dodecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic,margaric, stearic, acrylic, crotonic, undecylenic, oleic, hexynoic,heptynoic, octynoic acids, and the like; (b) saturated or unsaturated,alicyclic carboxylic acids, for example, cyclobutanecarboxylic acid,cyclopentanecarboxylic acid, cyclopentenecarboxylic acid,methylcyclopentenecarboxylic acid, cyclohexanecarboxylic acid,dimethylcyclohexanecarboxylic acid, dipropylcyclohexanecarboxylic acid,and the like; (c) saturated or unsaturated, alicyclic aliphaticcarboxylic acids, for example, cyclopentaneacetic acid,cyclopentanepropionic acid, cyclohexaneacetic acid, cyclohexanebutyricacid, methylcyclohexaneacetic acid, and the like; (d) aromaticcarboxylic acids, for example, benzoic acid, toluic acid, naphthoicacid, ethylbenzoic acid, isobutylbenzoic acid, methylbutylbenzoic acid,and the like; and (e) aromatic-aliphatic carboxylic acids, for example,phenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamicacid, phenylpropiolic acid and naphthylacetic acid, and the like.Suitable halo-, nitro-, hydroxy-, keto-, amino-, cyano-, thiocyano-, andlower alkoxyhydrocarbon carboxylic acids include hydrocarboncar-boxylicacids as given above which are substituted by one or more of halogen,nitro, hydroxy, keto, amino, cyano, or thiocyano, or loweralkoxy,advantageously loweralkoxy of not more than six carbon atoms, forexample, methoxy, ethoxy, propoxy, butoxy, amyloxy, hexyloxy, andisomeric forms thereof. Examples of such substituted hydrocarboncarboxylic acids are: mono-, di-, and trichloroacetic acid; α- andβ-chloropropionic acid; α- and γ-bromobutyric acid; α- and δ-iodovalericacid; mevalonic acid; 2- and 4-chlorocyclohexanecarboxylic acid;shikimic acid; 2-nitro-1-methyl-cyclobutanecarboxylic acid; 1, 2, 3, 4,5, 6-hexachlorocyclohexanecarboxylic acid;3-bromo-2-methylcyclohexanecarboxylic acid; 4- and5-bromo-2-methylcyclohexanecarboxylic acid; 5- and6-bromo-2-methylcyclohexanecarboxylic acid;2,5-dibromo-2-methylcyclohexanecarboxylic acid;4,5-dibromo-2-methylcyclohexanecarboxylic acid; 5,6-dibromo-2-methylcyclohexanecarboxylic acid;3-bromo-3-methylcyclohexanecarboxylic acid;6-bromo-3-methylcyclohexanecarboxylic acid;1,6-dibromo3-methylcyclohexanecarboxylic acid;2-bromo-4-methylcyclocohexanecarboxylic acid;1,2-dibromo-4-methylcyclohexanecarboxylic acid; 3-bromo-2, 2,3-trimethylcyclopentanecarboxylic acid;1-bromo-3,5-dimethylcyclohexanecarboxylic acid; homogentisic acid, o-,m-, and p-chlorobenzoic acid; anisic acid; salicylic acid;p-hydroxybenzoic acid; b-resorcylic acid; gallic acid; veratric acid;trimethoxybenzoic acid; trimethoxycinnamic acid; 4,4'-dichlorobenzilicacid; o-, m-, and p-nitrobenzoic acid; cyanoacetic acid; 3,4- and3,5-dinitrobenzoic acid; 2,4,6-trinitrobenzoic acid; thiocyanoaceticacid; cyanopropionic acid; lactic acid; ethoxyformic acid (ethylhydrogen carbonate); malic acid; citric acid; isocitric acid;6-methylsalicylic acid; mandelic acid; levulinic acid; pyruvic acid;glycine; alamine; valine; isoleucine; leucine; phenylalanine; proline;serine; threonine; tyrosine; hydroxyproline; ornithine; lysine;arginine; histidine; hydroxylysine; phenylglycine; p-aminobenzoic acid;m-aminobenzoic acid; anthranilic acid; aspartic acid; glutamic acid;aminoadipic acid; glutamine; asparagine; and the like.

7-Deoxynarciclasine Triacetate was prepared by reacting7-Deoxynarciclasine (0.5 g) with acetic anhydride (2 ml) and pyridine (2ml) at room temperature for 48 hours. Addition to ice water andfiltration gave a product (0.5 g) which was chromatographed on silicagel-60 (Merck; 70-230 mesh). Elution with methylene chloride-methanol(99:1) gave the triacetate (0.35 g) which crystallized from methylenechloride-methanol as colorless needles, mp 244°-246° [lit. 201° (9,11),233°-235° (12)]; [α]_(D) ²⁷ +219° (c 1.0, CHCl₃) [lit. [α]²⁰ +195° (c0.45, CHCl₃)(11)]; eims m/e 4.17 (M⁺ C₂₀ H₁₉ NO₉); λ_(max)^(CH).sbsp.3^(OH) (log Σ), 231(4.20), 251(4.21) and 305(3.93) nm; ir(KBr) ν_(max) 3392, 1760, 1748, 1733, 1663, 1640, 1615, 1500, 1485,1470, 1400, 1373, 1364, 1263, 1245, 1225, 1076, 1040, 1015, 966, 940,828 and 663 cm⁻¹ ; ¹ H nmr (CDCl₃) δ 2.12 (3H, s), 2.14 (3H, s), 2.19(3H, s), 4.71 (1H, dd, J=9 and 2Hz), 5.32 (1H, dd, J=9 and 2Hz), 5.38(1H, m), 5.55 (1H, m), 6.12 (1H, m), 6.12 (2H, s), 7.06 (1H, s), 7.24(1H, br. s, removed by D₂ O) and 7.59 (1H, s) ppm; ¹³ C nmr (CDCl₃) δ170.4, 169.79, 169.53, 164.52, 151.79, 149.25, 134.24, 130.43, 122.56,117.12, 107.52, 103.43, 102.07, 71.23, 68.59, 68.30, 50.27, 21.02, 20.86and 20.73 ppm.

Anal calcd for C₂₀ H₁₉ NO₉ : C, 57.55; H, 4.55. Found: C, 57.37; H,4.63.

The structure was solved by direct methods and atomic co-ordinatesrefined by full matrix least-squares programs provided in a structuresolution package. Hydrogen atom co-ordinates were either calculatedand/or located via difference maps and were included in the finalrefinement. The final standard crystallographic residuals (weighted andunweighted R factors) for the model, which contained anisotropictemperature factors for all heavy atoms and fixed (B=4.0) isotropicfactors for all hydrogens, were 0.063 and 0.044, respectively. Themaximum shift to error ratio in the last cycle of refinement was 0.55,with all bond distances and angles having nominal values.

Crystal data: C₂₀ H₁₉ NO₉, monoclinic, space group P2₁, with a=8.325(2),b=8.013(2), c=14.551(2)Å, V=946.4Å³, Dm=1.45, Dc=1.46 g cm⁻³ for Z=2.One quadrant of data on a crystal of dimensions ca. 0.10×0.15×0.75 mmwas collected to a maximum of 2θ of 150° using the w/2θ scan techniqueand graphite monochromated Cu Kα radiation (λ 1.5418Å), and afterLorentz and polarization corrections, 1581 of the reflections with /F/≧3σ (F) were used in the structure determination; absorbtion correctionswere deemed unecessary (μ=9.5 cm⁻¹).

Pancratistatin Pentaacetate was prepared when pancratistatin (0.5 g) wastreated in a similar manner to that described above for7-deoxynarciclasine. The product (0.5 g) was chromatographed onSephadex® LH-20 (100 g) using methanol-methylene chloride (3:2) aseluent to give the amorphous pentaacetate, mp 162°-166°; [α]²⁹ +85° (c1.0, CHCl₃); eims m/e 535 (M⁺,C₂₄ H₂₅ NO₁₃); λ_(max) ^(CH).sbsp.3^(OH)(log Σ) 227(4.31), 247(sh), 271(sh) and 299(3.75 nm); ir (KBr) ν_(max)3370, 1760, 1680, 1635, 1510, 1490, 1375, 1340, 1295, 1250, 1220, 1180,1080, 1045, 950, 930, 860, 815, 758 and 640 cm⁻¹ ; ¹ H nmr (90 MHz;CDCl₃) δ 2.06 (6H, s), 2.08 (3H, s), 2.17 (3H, s), 2.37 (3H, s), 3.43(1H, dd, J=12.3 and 2.5 Hz), 4.26 (1H, dd, J=12.5 and 11.7 Hz), 5.14(1H, dd, J=11.7 and 3.3 Hz), 5.22 (1H, dd, J=2.9 and 2.9 Hz), 5.45 (1H,m), 5.56 (1H, m), 5.76 (1H, br. s, removed by D₂ 0), 6.08 (2H, br. s)and 6.48 (1H, s) ppm; ¹³ C nmr (CDCl₃) δ 170.08, 169.69, 169.07, 169.01,168.29, 162.96, 152.60, 139.86, 134.53, 132.90, 116.20, 102.94, 101.84,71.68, 67.69, 66.84, 66.42, 47.86, 40.00, 20.86 and 20.70 (last twosignals 5 C) ppm.

Anal calcd for C₂₄ H₂₅ NO₁₃ :C, 53.83; H, 4.67; N, 2.62. Found: C,53.75; H, 4.68; N, 2.61.

Pancratistatin Methyl Ether was prepared by reacting pancratistatin(0.33 g) in methanol (100 ml) with excess diazomethane in ether. Afterstirring for 8 hours at room temperature an additional quantity ofdiazomethane was added and stirring continued 8 hours. Evaporation ofthe solvent gave a product (0.34 g) which was chromatographed onSephadex® Sephadex® LH-20 using methanol as eluent to give the methylether (0.1 g). Crystallization from methanol gave colorless plates; mp294°-298° (dec); [α]_(D) ²⁵ +289.9° (c 0.69, DMSO); ir (KBr) ν_(max)3500, 3400, 3300, 1635, 1600, 1485, 1450, 1390, 1343, 1298, 1226, 1207,1152, 1122, 1090, 1060, 1035, 967, 937, 920, 880, 840, 795, 724, 660 and620 cm⁻¹ ; ¹ H nmr (DMSO-d₆) δ 3.58-4.44 (6H), 3.88 (3H, s), 4.70-5.50(4H, removed by D₂ O), 6.13 (2H, d, J=5 Hz), 6.74 (1H, s) and 6.92 (1H,removed by D₂ O) ppm.

Anal calcd for C₁₅ H₁₇ NO₈ : C, 53.10; H, 5.01; N, 4.13. Found: C,53.44; H, 5.06; N, 4.08.

The administration of pancratistatin and its pharmacologically activephysiologicaly compatible derivatives is useful for treating animals orhumans bearing a neoplastic disease, for example, acute myelocyticleukemia, acute lymphocytic leukemia, malignant melanoma, adenocarcinomaof lung, neuroblastoma, small cell carcinoma of lung, breast carcinoma,colon carcinoma, ovarian carcinoma, bladder carcinoma, and the like.

The dosage administered will be dependent upon the identity of theneoplastic disease; the type of host involved, including its age, healthand weight; the kind of concurrent treatment, if any; the frequency oftreatment and therapeutic ratio.

Illustratively, dosage levels of the administered active ingredientsare: intravenous, 0.1 to about 200 mg/kg; intramuscular, 1 to about 500mg/kg; orally, 5 to about 1000 mg/kg; intranasal instillation, 5 toabout 1000 mg/kg; and aerosol, 5 to about 1000 mg/kg of host bodyweight.

Expressed in terms of concentration, an active ingredient can be presentin the compositions of the present invention for localized use about thecutis, intranasally, pharyngolaryngeally, bronchially, broncholially,intravaginally, rectally, or ocularly in a concentration of from about0.01 to about 50% w/w of the composition; preferably about 1 to about20% w/w of the composition; and for parenteral use in a concentration offrom about 0.05 to about 50% w/v of the composition and preferably fromabout 5 to about 20% w/v.

The compositions of the present invention are preferably presented foradministration to humans and animals in unit dosage forms, such astablets, capsules, pills, powders, granules, suppositories, sterileparenteral solutions or suspensions, sterile non-parenteral solutions orsuspensions, and oral solutions or suspensions and the like, containingsuitable quantities of an active ingredient.

For oral administration either solid or fluid unit dosage forms can beprepared.

Powders are prepared quite simply by comminuting the active ingredientto a suitably fine size and mixing with a similarly comminuted diluent.The diluent can be an edible carbohydrate material such as lactose orstarch. Advantageously, a sweetening agent or sugar is present as wellas a flavoring oil.

Capsules are produced by preparing a powder mixture as hereinbeforedescribed and filling into formed gelatin sheaths. Advantageously, as anadjuvant to the filling operation, a lubricant such as a talc, magnesiumsterate, calcium stearate and the like is added to the powder mixturebefore the filling operation.

Soft gelatin capsules are prepared by machine encapsulation of a slurryof active ingredients with an acceptable vegetable oil, light liquidpetrolatum or other inert oil or triglyceride.

Tablets are made by preparing a powder mixture, granulating or slugging,adding a lubricant and pressing into tablets. The powder mixture isprepared by mixing an active ingredient, suitably comminuted, with adiluent or base such as starch, lactose, kaolin, dicalcium phosphate andthe like. The powder mixture can be granulated by wetting with a bindersuch as corn syrup, gelatin solution, methylcellulose solution or acaciamucilage and forcing through a screen. As an alternative to granulating,the powder mixture can be slugged, i.e., run through the tablet machineand the resulting imperfectly formed tablets broken into pieces (slugs).The slugs can be lubricated to prevent sticking to the tablet-formingdies by means of the addition of stearic acid, a stearic salt, talc ormineral oil. The lubricated mixture is then compressed into tablets.

Advantageously the tablet can be provided with a protective coatingconsisting of a sealing coat or enteric coat of shellac, a coating ofsugar and methylcellulose and polish coating of carnauba wax.

Fluid unit dosage forms for oral administration such in syrups, elixirsand suspensions can be prepared wherein each teaspoonful of compositioncontains a predetermined amount of active ingredient for administation.The water-soluble forms can be dissolved in an aqueous vehicle togetherwith sugar, flavoring agents and preservatives to form a syrup. Anelixir is prepared by using a hydroalcoholic vehicle with suitablesweeteners together with a flavoring agent. Suspensions can be preparedof the insoluble forms with a suitable vehicle with the aid of asuspending agent such as acacia, tragacanth, methylcellulose and thelike.

For parenteral administration, fluid unit dosage forms are preparedutilizing an active ingredient and a sterile vehicle, water beingpreferred. The active ingredient, depending on the form andconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the water-soluble active ingredient can bedissolved in water for injection and filter sterilized before fillinginto a suitable vial or ampule and sealing. Advantageously, adjuvantssuch as a local anesthetic, preservative and buffering agents can bedissolved in the vehicle. Parenteral suspensions are prepared insubstantially the same manner except that an active ingredient issuspended in the vehicle instead of being dissolved and sterilizationcannot be accomplished by filtration. The active ingredient can besterilized by exposure to ethylene oxide before suspending in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of theactive ingredient.

In addition to oral and parenteral administration, the rectal andvaginal routes can be utilized. An active ingredient can be administeredby means of a suppository. A vehicle which has a melting point at aboutbody temperature or one that is readily soluble can be utilized. Forexample, cocoa butter and various polyethylene glycols (Carbowaxes) canserve as the vehicle.

For intranasal instillation, a fluid unit dosage form is preparedutilizing an active ingredient and a suitable pharmaceutical vehicle,preferrably P.F. water, a dry powder can be formulated when insufflationis the administration of choice.

For use as aerosols, the active ingredients can be packaged in apressurized aerosol container together with a gaseous or liquifiedpropellant, for example, dichlorodifluormethane, carbon dioxide,nitrogen, propane, and the like, with the usual adjuvants such ascosolvents and wetting agents, as may be necessary or desirable.

The term "unit dosage form" as used in the specification and claimsrefers to physically discrete units suitable as unitary dosages forhuman and animal subjects, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required pharmaceutical diluent, carrier orvehicle. The specifications for the novel unit dosage forms of thisinvention are dictated by and are directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitation inherent in the art ofcompounding such an active material for therapeutic use in humans, asdisclosed in this specification, these being features of the presentinvention. Examples of suitable unit dosage forms in accord with thisinvention are tablets, capsules, troches, suppositories, powder packets,wafers, cachets, teaspoonfuls, tablespoonfuls, dropperfuls, ampules,vials, segregated multiples of any of the foregoing, and other forms asherein described.

The active ingredients to be employed as antineoplastic agents can beeasily prepared in such unit dosage form with the employment ofpharmaceutical materials which themselves are available in the art andcan be prepared by established procedures. The following preparationsare illustrative of the preparation of the unit dosage forms of thepresent invention, and not as a limitation thereof.

EXAMPLE 1

Several dosage forms were prepared embodying the present invention. Theyare shown in the following examples in which the notation "activeingredient" signifies pancratistatin and 7-deoxynarciclasine, theirsynthetic counterparts and the non-toxic pharmaceutically activederivatives thereof.

COMPOSITION "A" Hard-Gelatin Capsules

One thousand two-piece hard gelation capsules for oral use, each capsulecontaining 200 mg of an active ingredient are prepared from thefollowing types and amounts of ingredients:

    ______________________________________                                        Active ingredient, micronized                                                                         200    gm                                             Corn Starch             20     gm                                             Talc                    20     gm                                             Magnesium stearate      2      gm                                             ______________________________________                                    

The active ingredient, finely divided by means of an air micronizer, isadded to the other finely powdered ingredients, mixed thoroughly andthen encapsulated in the usual manner.

The foregoing capsules are useful for treating a neoplastic disease bythe oral administration of one or two capsules one to four times a day.

Using the procedure above, capsules are similarly prepared containing aactive ingredient in 50, 250 and 500 mg amounts by substituting 50 gm,250 gm and 500 gm of a active ingredient for the 200 gm used above.

COMPOSITION "B" Soft Gelatin Capsules

One-piece soft gelatin capsules for oral use, each containing 200 mg ofa active ingredient (finely divided by means of an air micronizer), areprepared by first suspending the compound in 0.5 ml of corn oil torender the material capsulatable and then capsulating in the abovemanner.

The foregoing capsules are useful for treating a neoplastic disease bythe oral administration of one or two capsules one to four times a day.

COMPOSITION "C" Tablets

One thousand tablets, each containing 200 mg of a active ingredient areprepared from the following types and amounts of ingredients:

    ______________________________________                                        Active ingredient micronized                                                                          200    gm                                             Lactose                 300    gm                                             Corn starch             50     gm                                             Magnesium stearate      4      gm                                             Light liquid petrolatum 5      gm                                             ______________________________________                                    

The active ingredient finely divided by means of an air micronizer, isadded to the other ingredients and then thoroughly mixed and slugged.The slugs are broken down by forcing through a Number Sixteen screen.The resulting granules are then compressed into tablets, each tabletcontaining 200 mg of the active ingredient.

The foregoing tablets are useful for treating a neo-plastic disease bythe oral administration of one or two tablets one to four times a day.

Using the procedure above, tablets are similarly prepared containing aactive ingredient in 250 mg and 100 mg amounts by substituting 250 gmand 100 gm of a active ingredient for the 200 gm used above.

COMPOSITION "D" Oral Suspension

One thousand ml of an aqueous suspension for oral use, containing ineach teaspoonful (5 ml) dose, 50 mg of a active ingredient, is preparedfrom the following types and amounts of ingredients:

    ______________________________________                                        Active ingredient micronized                                                                          10     gm                                             Citric acid             2      gm                                             Benzoic acid            1      gm                                             Sucrose                 790    gm                                             Tragacanth              5      gm                                             Lemon Oil               2      gm                                             Deionized water, q.s. 1000 ml.                                                ______________________________________                                    

The citric acid, benzoic acid, sucrose, tragacanth and lemon oil aredispersed in sufficient water to make 850 ml of suspension. The activeingredient finely divided by means of an air micronizer, is stirred intothe syrup until uniformly distributed. Sufficient water is added to make1000 ml.

The composition so prepared is useful for treating a neoplastic diseaseat a dose of 1 tablespoonful (15 ml) three times a day.

COMPOSITION "E" Parenteral Product

A sterile aqueous suspension for parenteral injection, containing in 1ml 300 mg of a active ingredient for treating a neoplastic disease, isprepared from the following types and amounts of ingredients:

    ______________________________________                                        Active ingredient, micronized                                                                          30     gm                                            Polysorbate 80           5      gm                                            Methylparaben            2.5    gm                                            Propylparaben            0.17   gm                                            Water for injection, q.s. 1000 ml.                                            ______________________________________                                    

All the ingredients, except the active ingredient, are dissolved in thewater and the solution sterilized by filtration. To the sterile solutionis added the sterilized active ingredient, finely divided by means of anair micronizer, and the final suspension is filled into sterile vialsand the vials sealed.

The composition so prepared is useful for treating a neoplastic diseaseat a dose of 1 milliliter (1M) three times a day.

COMPOSITION "F" Suppository, Rectal and Vaginal

One thousand suppositories, each weighing 2.5 gm and containing 200 mgof a active ingredient are prepared from the following types and amountsof ingredients:

    ______________________________________                                        Active ingredient, micronized                                                                         15     gm                                             Propylene glycol        150    gm                                             Polyethylene glycol #4000, q.s.                                                                       2,500  gm                                             ______________________________________                                    

The active ingredient is finely divided by means of an air micronizerand added to the propylene glycol and the mixture passed through acolloid mill until uniformly dispersed. The polyethylene glycol ismelted and the propylene glycol dispersion added slowly with stirring.The suspension is poured into unchilled molds at 40°C. The compositionis allowed to cool and solidify and then removed from the mold and eachsuppository foil wrapped.

The foregoing suppositories are inserted rectally or vaginally fortreating a neoplastic disease.

COMPOSITION "G" Intranasal Suspension

One thousand ml of a sterile aqueous suspension for intranasalinstillation, containing in each ml 200 mg of a active ingredient, isprepared from the following types and amounts of ingredients:

    ______________________________________                                        Active ingredient, micronized                                                                         15     gm                                             Polysorbate 80          5      gm                                             Methylparaben           2.5    gm                                             Propylparaben           0.17   gm                                             Deionized water, q.s. 1000 ml.                                                ______________________________________                                    

All the ingredients, except the active ingredient, are dissolved in thewater and the solution sterilized by filtration. To the sterile solutionis added the sterilized acitve ingredient, finely divided by means of anair micronizer, and the final suspension is aseptically filed intosterile containers.

The composition so prepared is useful for treating a neoplastic disease,by intranasal instillation of 0.2 to 0.5 ml given one to four times perday.

An active ingredient can also be present, as shown in Examples 12-14 inthe undiluted pure form for use locally about the cutis, intranasally,pharyngolaryngeally, bronchially, broncholially or orally.

COMPOSITION "H" Power

Five grams of a active ingredient in bulk form is finely divided bymeans of an air micronizer. The micronized power is placed in ashaker-type container.

The foregoing composition is useful for treating a neoplastic disease,at localized sites by applying a powder one to four times per day.

COMPOSITION "I" Oral Powder

One hundred grams of a active ingredient in bulk form is finely dividedby means of an air micronizer. The micronized powder is divided intoindividual doses of 200 mg and packaged.

The foregoing powders are useful for treating a neoplastic disease, bythe oral administration of one or two powders suspended in a glass ofwater, one to four times per day.

COMPOSITION "J" Insufflation

One hundred grams of a active ingredient in bulk form is finely dividedby means of an air micronizer.

The foregoing composition is useful for treating a neoplastic disease,by the inhalation of 300 mg one to four times per day.

COMPOSITION "K" Hard Gelatin Capsules

One hundred two-piece hard gelatin capsules for oral use, each capsulecontaining 200 mg of a active ingredient.

The active ingredient is finely divided by means of an air micronizerand encapsulated in the usual manner.

The foregoing capsules are useful for treating a neoplastic disease, bythe oral administration of one or two capsules, one to four times a day.

Using the procedure above, capsules are similarly prepared containingactive ingredient in 50, 250 and 500 mg amounts by substituting 50 gm,250 gm and 500 of the active ingredient for the 200 gm used above.

EXAMPLE 2

Unit dosage forms of pancratistatin prepared according to selectedcompositions described in Example 1 were screened utilizing Protocol1,200 described in Cancer Chemotherapy Reports, part 3, Vol. 3, No. 2,Sept. 1972, pp 9 et seq for lymphocytic leukemia P388. Pancratistatinprovided a 38-106% life extension at 0.75-12.5 mg/Kg host body weightagainst the murine P388 lymphocytic leukemia. Pancratistatin alsomarkedly inhibited growth of the P388 in vitro cell line (ED₅₀, 0.001μg/ml).

EXAMPLE 3

Unit dosage forms of 7-deoxynarciclasine were prepared according toExample 1 and were screened using accepted protocols of the NationalCancer Institute. The preparation obtained up to 61% life extension at12 mg per kg host body weight against murine P388 lympocytic leukemiaand an ED 50 of 0.02 micrograms per ml against the P388 cell line.

EXAMPLE 4

A unit dosage form of pancratistatin, prepared according to Example 1,was challenged with M5074 murine ovary sarcoma and obtained a 53% to 84%life extension at 0.38 to 3.0 mg active reagent/Kg host body weightusing the National Cancer Institute accepted protocol for lifeextension.

EXAMPLE 5

A unit dosage form of pancratistatin, prepared according to Example 1,was subjected to the National Cancer Institute accepted protocol forcure rate against its M5074 murine ovary sarcoma and obtained a 50% curerate at 6 mg/Kg host body weight.

From the foregoing it becomes readily apparent that a new an usefulantineoplastic factor and new and useful antineoplastic preparationshave been herein described and illustrated which fulfill all of theaforestated objectives in a remarkably unexpected fashion. It is ofcourse understood that such modifications, alterations and adaptationsas will readily occur to the artisan confronted with this disclosure areintended within the spirit of the present invention which is limitedonly by the scope of the claims appended hereto.

Accoridngly, what is claimed is:
 1. An antineoplastic substancedenominated pancratistatin.
 2. An antineoplastic substance having thestructural formula: ##STR7## wherein: R=H, CH₃ or COX;R₁ =H or COX; R₂=H or COX; R₃ =H or COX; R₄ =H or COX; and X⁴ =a radical of alkyl(C--1--20); alkenyl (C=1--20), aryl (C=9--12), or heterocyclic selectedfrom the group consisting of thiazoyl, thiophenoyl, pyridoyl, pyrimidoyland furoyl.
 3. An antineoplastic substance according to claim 2 whereinR=H and R₁ =R₂ =R₃ =R₄ =H.
 4. An antineoplastic pharmaceuticalpreparation containing as its principal active ingredients an effectiveamount of natural or synthetic substances or pharmaceutically activederivatives thereof a first substance having the structure ##STR8##wherein: R=H, CH₃ or COX;R₁ =H or COX; R₂ =H or COX; R₃ =H or COX; R₄ =Hor COX; and X⁴ =a radical of alkyl (C--1--20), alkenyl (C=1--20), aryl(C=9--12), or heterocyclic selected from the group consisting ofthiazoyl, thiophenoyl, pyridoyl, pyrimidoyl and furoyl;and a secondsubstance having the structure "B" ##STR9## wherein: R₁ =H, COOH₃ orCOX; R₂ =H, COCH₃ or COX; R₃ =H, COCH₃ or COX; and X³ =a radical ofalkyl (C--1--20); alkenyl (C=1--20), aryl (C=9--12), or heterocyclicselected from the group consisting of thiazoyl, thiophenoyl, pyridoyl,pyrimidoyl and furoyl.
 5. An antineoplastic pharmaceutical preparationaccording to claim 4 in which said substance has the "A" structurewherein: R=R₁ =R₂ =R₃ =H.
 6. An antineoplastic preparation according toclaim 4 in which said second substance has the "B" structure wherein:R=H and R₁ =R₂ =R₃ =H.
 7. An antineoplastic pharmaceutical preparationaccording to claim 5 having the "B" structure wherein: R=H and R₁ =R₂=R₃ =H.
 8. A substance according to claim 2 in which said COX has thestructure: ##STR10## wherein: n=0-2.
 9. A substance according to claim 2in which said COX has the structure: ##STR11## wherein: n=0 to
 19. 10. Asubstance according to claim 2 in which said COX has the structure:##STR12## wherein: m=0-3p=0-16